The overall goal of this project is to identify small molecule inhibitors of HIV Vif that inhibit binding to its cellular cofactor APOBEC3G (A3G) and other APOBEC3 proteins. Vif is required for HIV replication, and is therefore a potential therapeutic target. A3G and the related cytidine deaminase APOBEC3F (A3F) inhibit HIV replication by inducing G to A hypermutation in viral DNA, in addition to deamination-independent mechanisms. Vif overcomes the innate antiviral activity of A3G and A3F by binding these cellular proteins and targeting them for proteasomal degradation. Currently, there are no antivirals that target Vif or its interactions with host cofactors. In preliminary studies, we developed a high-throughput screen (HTS) in 384-well format to identify inhibitors of Vif-A3G binding. We validated the HTS by automated screening of 2,640 bioactive compounds, which demonstrated that the screen is specific and robust and identified 15 compounds that are being evaluated for their ability to inhibit Vif function and viral replication in cell-based assays. Aim 1 will use this HTS to complete screening of 100,000 compounds at the Harvard ICCB to identify small molecule inhibitors of Vif-A3G binding and test the best hits in secondary screens to identify those that specifically inhibit Vif-A3G binding, Vif- mediated degradation of A3G, and HIV replication. Aim 2 will select for drug-resistant HIV strains that develop resistance to the best hits identified in Aim 1 during passage ex vivo and determine if resistance mutations localize to the vif gene. Aim 3 will perform antiviral efficacy studies in a humanized mouse model to determine whether lead compounds inhibit HIV replication in vivo. These studies are expected to identify small molecule Vif inhibitors that inhibit Vif binding to A3G and other APOBEC3 proteins, and may identify novel lead compounds with potential for further development as new anti-viral therapies. PUBLIC HEALTH RELEVANCE: This project will use a new high throughput screening assay to identify inhibitors of the HIV Vif protein that inhibit Vif binding to its host cell targets APOBEC3G and APOBEC3F. These studies may identify small molecules that inhibit HIV Vif, including lead compounds that could be further developed as new anti-viral therapies